The objectives of this project, and related progress in the past year, are reviewed below. &#8232;&#8232; (1) Development of a reverse genetics system that can be used to modify the antigenicity and virulence of rotaviruses (RVs) and to develop new vaccine candidates. To expand the usefulness of RV reverse genetics, we developed a novel method to recover single-gene recombinant viruses in which two independent mechanisms act on a single viral gene to select for its replacement with a gene of recombinant origin. Specifically, we have used a mutant RV bearing a temperature-sensitive (ts) defect in the viral NSP2 protein with RNAi-mediated degradation of NSP2 mRNAs to isolate a virus containing a single recombinant gene that evades both selection mechanisms. Recovery of recombinant viruses by this system is rapid and highly efficient. We published a paper describing this reverse genetics system in October 2010 (Trask et al, PNAS USA 107: 18652-7). In the past year, we also initiated experiments using the reverse genetics system designed to test the possibility that the RV genome could be manipulated in a way that allows the generation of RVs that express foreign proteins. By this approach, it might be possible to create RV vaccines that could induce protection against not only RV diarrheal disease, but also against other diseases (e.g., HCV, HIV). So far, our analyses have shown that we can create genetically-stable recombinant RVs that contain extensive stretches of non-rotaviral sequences, such as HCV sequences, and a cap-independent ribosome entry site sequence (IRES). (2) Identification of RV virulence determinants that antagonize host immune responses, notably the interferon signaling pathways. Studies involving limited numbers of rotavirus (RV) strains have shown that the viral gene 5 product, NSP1, can antagonize beta interferon (IFN-&#946;) expression by inducing the degradation of IFN-regulatory factors (IRFs) (IRF3, IRF5, and IRF7) or a component of the E3 ubiquitin ligase complex responsible for activating NF-&#954;B (&#946;-transducin repeat-containing protein &#946;-TrCP). To gain a broader perspective of NSP1 activities, we examined various RV strains for the ability to inhibit IFN-&#946;expression in human cells. We found that all strains encoding wild-type NSP1 impeded IFN-&#946;expression but not always through IRF3 degradation. To identify other degradation targets involved in suppressing IFN-&#946;expression, we used transient expression vectors to test the abilities of a diverse collection of NSP1 proteins to target IRF3, IRF5, IRF7, and &#946;-TrCP for degradation. The results indicated that human RVs rely predominantly on the NSP1-induced degradation of IRF5 and IRF7 to suppress IFN signaling, whereas NSP1 proteins of animal RVs tended to target IRF3, IRF5, and IRF7, allowing the animal viruses a broader attack on the IFN-&#946;signaling pathway. The results also suggested that the NSP1-induced degradation of &#946;-TrCP is an uncommon mechanism of subverting IFN-&#946;signaling but is one that can be shared with NSP1 proteins that induce IRF degradation. Our analysis reveals that the activities of NSP1 proteins are diverse, with no obvious correlations between degradations of pairs of target proteins. Thus, RVs have evolved functionally distinct approaches for subverting the host antiviral response, a property consistent with the immense sequence variation noted for NSP1 proteins. The results of this study were published in March 2011 (Arnold and Patton, J Virol 85: 1970-90). In the past year, we also initiated studies aimed at mapping the interaction domain in IRF proteins that is recognized by NSP1. The results indicate that NSP1 binds to the dimerization domain in IRF proteins. (3) Analysis of the diversity and evolution of the RV genome. (a) An important question is whether the widespread use of the RotaTeq and Rotarix vaccines will induce antigenic and/or genetic changes in commonly circulating RVs or will lead to the emergence of new G-type strains not covered by the vaccines. To address this question, we initiated a project to sequence the complete genomes of RVs collected from sick children at Vanderbilt Medical Center during the 2005-06 to 2008-09 winter seasons. The four seasons covered by these samples are particularly significant as they include seasons prior to and after introduction of the RotaTeq vaccine. In spring of 2011, sequencing of the genomes of the RVs collected at Vanderbilt was completed;the results are currently undergoing statistical analysis. (b) To gain insight into the diversity and evolution of human RVs circulating in a single location over a long period of time, we are in the process of analyzing the genomic sequences of G1, G2, G3, and G4 RVs isolated from sick children during 1974 to 1991 at Children's Hospital National Medical Center, Washington DC. Sequencing of the G2, G3, and G4 samples have been completed. The G3 results were published in 2009 (McDonald et al, PLoS Pathog 5:e1000634) and the G4 results were published in June 2011 (McDonald et al, Infect Genet Evol, epub). Sequencing of the G1 samples is ongoing, but should be completed by spring 2012. To date, the results of our analysis have indicated that genetically-distinct RV clades of the same G/P-type, but with varying neutralization epitopes, can co-circulate and cause disease within a single geographical region. The findings also suggest that although genome reassortment can occur among RVs, most reassortant strains are replaced overtime by lineages with preferred gene constellations. (4) Analysis of the genetic make-up and stability of RV vaccine candidates. Dr. Albert Kapikian and colleagues developed virus strains for a multivalent RV vaccine (UK BRV) by reassortment of antigenically-distinct human RVs with the bovine UK RV. Eight reassortant strains were created, differing only in their VP7 component and their VP7 serotype (G1, G2, G3, G4, G8, G9, G10, G12). Master seed lots were prepared for each reassortant by the NIH (NIH Master Seeds) and distributed to companies interested in producing RV vaccines at low cost in developing counties. As part of their program to develop the vaccine, three companies have generated their own Master Seeds (Shantha, India;Sinovac Biotech, China;Wuhan Institute of Biological Products (WIBP), China) from the NIH Master Seeds, and then used their Master Seeds subsequently to produce Working Batches and Production Batches of each viral component of the UK BRV vaccine. To support development of RV vaccines, we have agreed to sequence the viral genomes in the Master Seeds and the Working and Production Batches produced by the companies. This information is necessary to provide safety information to regulatory agencies concerning the genetic make-up and stability of viruses in RV vaccines. In the past year, we completed sequencing efforts on WIBP Master Seeds, and Working and Production Batches. Analysis of the sequences obtained for the WIBP samples (156 genes) indicates that the vaccine virus strains are genetically stable. The sequences have been deposited in GenBank.